Lecture Enzymes
-
Upload
john-alfrey-dondiego-pueblo -
Category
Documents
-
view
217 -
download
0
Transcript of Lecture Enzymes
-
7/28/2019 Lecture Enzymes
1/45
ENZYMES
Siv Milliscent E. Balbas, RMT
-
7/28/2019 Lecture Enzymes
2/45
What are Enzymes?
CHON catalyst of chemical reactions andcatalyzes all metabolic reactions of cells
(eg. Respiration, photosynthesis, digestion,)
biological catalyst
specific for its substrate
-
7/28/2019 Lecture Enzymes
3/45
Parts of an Enzyme
Active site- portion of the enzyme wherereaction takes place
Substrate- binds to Active site of an enzyme
Allosteric site Other site besides the active site
-
7/28/2019 Lecture Enzymes
4/45
ENZYME
SUBSRTATE
LOCK AND KEY FIT
-
7/28/2019 Lecture Enzymes
5/45
ENZYME
SUBSRTATE
INDUCED FIT
-
7/28/2019 Lecture Enzymes
6/45
Cofactor
Nonprotein molecule which maybe necessary forenzyme activity
Coenzyme- Organic cofactor (NAD, heme,
vitamins)
Activators- Inorganic cofactor (eg. Chloride or
magnesium ions, and other metals)
-
7/28/2019 Lecture Enzymes
7/45
ENZYME
SUBSRTATE
Cofactor
HOLOENZYME
APOENZYME
-
7/28/2019 Lecture Enzymes
8/45
NOTE:
Holoenzyme- complete, active enzyme system Proenzyme (zymogen)- inactive enzyme
Isoenzyme- enzymes that have differentphysical properties but have the same
catalytic functions
-
7/28/2019 Lecture Enzymes
9/45
Classification of Enzymes
OXIDOREDUCTASES
Catalyze an oxidation-reduction reaction between2 substrates
TRANSFERASES
Catalyze the transferof a group from one
substrate to another
-
7/28/2019 Lecture Enzymes
10/45
HYDROLASES
Catalyzes hydrolysis of various bonds
LYASES
Removalof groups without hydrolysis
ISOMERASES Catalyze the interconversion of geometric, optical
or positional isomers
LIGASES Catalyze the joining of two substrate molecules,
-
7/28/2019 Lecture Enzymes
11/45
Factors the Influence
Enzymatic Reactions SUBSTRATE CONCENTRATION
pH ( 7.0- 8.0)
TEMPERATURE
ENZYME CONCENTRATION
COFACTORS
INHIBOTORS Competitive inhibitors
Noncompetitive inhibitor
Uncompetitive inhibitor
-
7/28/2019 Lecture Enzymes
12/45
Competitive Inhibitors
Competes with substrate for binding to active site
Non-Competitive Inhibitors
Does not bind to the active site, but removes
cofactors of the enzyme
Uncompetitive Inhibitors
Inhibitor binds to E-S complexpreventingdissociation
-
7/28/2019 Lecture Enzymes
13/45
Enzyme Tissue Specificity
High Moderate Low
ACP(RBC, Prostate)
AST(Liver, heart, skeletal
muscle))
LD(In ALL tissues)
ALT(Liver)
CK(Heart, skeletal muscle,
brain)
AMS(Pancreas, salivary glands) ALP(liver, bone, kidney)
LPS(Pancreas)
-
7/28/2019 Lecture Enzymes
14/45
Hepatic Enzyme Profile
ALP
AST
LD (LDH)
GGT
ChE
-
7/28/2019 Lecture Enzymes
15/45
Alkaline Phosphatase (ALP)
optimum pH : 10
highest concentration in liver, kidney,placenta, intestine, WBC
-
7/28/2019 Lecture Enzymes
16/45
Increase levels in:
Bone formation
Pagets disease
Rickets
Osteoblastic tumors
Cancer
Sever fracture
Liver disease
-
7/28/2019 Lecture Enzymes
17/45
Collection:
AVOID using citrate, oxalate, EDTA
Substrate: Organic phosphates (eg. -glyceroPO4 and -nitrophenylPO4)
Methods:
Bodansky(-glyceroPO4)
Shenowara Jones (-glyceroPO4) King- Armstrong (-nitrophenylPO4)
Bessy Lowry-Brock(-nitrophenylPO4)
-
7/28/2019 Lecture Enzymes
18/45
Alkaline AminoTransferase
(ALT) Serum Glutamic PyruvicTransaminase (SGPT) Found in the LIVER and RBCs
More specific for liver than AST
Marked elevation with viral hepatitis
Collection: AVOID HEMOLYSIS
-
7/28/2019 Lecture Enzymes
19/45
Catalyzes the reaction:
Alanine + ketoglutaric acid Pyruvic Acid
Method:
Reitman Frankel
Addition of DNPH (2,4- dihydrophenylhydrazine) topyruvic acid to produce color
ALT
-
7/28/2019 Lecture Enzymes
20/45
ALT (Liver)
AST (Heart)Glutamate
oxalacetatetransaminase
Substrate Alanine - Keto Aspartate keto
End Product Pyruvic Acid Oxaloacetic Acid
Location Heart, LIVER HEART, Liver
HighConcentration
LIVER HEART
Significance Liver DiseaseMycocardial
Infarction
-
7/28/2019 Lecture Enzymes
21/45
ALT/AST RATIOde ritis ratio
Differentiates VIRAL from NON-VIRAL etiology
Viral Etiology: High ALT If ALT/AST is >1 = VIRAL
Non-viral: High AST If ALT/AST is
-
7/28/2019 Lecture Enzymes
22/45
Lactate DeHydrogenase
(LD/LDH)
Catalyzes reversible lactate pyruvate
using NAD+
as a cofactor
5 isoenzymes (electrophoresis):
LD1, LD2, LD3, LD4, LD5
-
7/28/2019 Lecture Enzymes
23/45
4 SUBUNITS in each Isoenzyme
ISOENZYME SUBUNITS SHORTHANDSUBUNITS
HIGHCONCENTRATION
LD1 HHHH LD H4 Heart, RBC, Brain
LD2 HHHM LD H3M Heart, RBC, Brain
LD3 HHMM LD H2M2Brain, Kidney,
Lung
LD4 HMMM LD HM3 Liver, Skeletalmuscle, kidney
LD5 MMMM LD M4Liver, Skeletalmuscle, ileum
-
7/28/2019 Lecture Enzymes
24/45
Collection: AVOID HEMOLYSIS
clot must be separated from serum
quickly to prevent increase in LD1 and LD2
Storage: 25C upto 24 hours
Only LD4 and LD5 are utilized to test LIVERDISORDERS. May also be elevated duringskeletal muscle damage
-
7/28/2019 Lecture Enzymes
25/45
Gamma-Glutamyl
Transferase/Transpeptidase
(GGT)
Increase in liver disease, metastaticcarcinoma , alcoholism and hepatobiliary
obstruction
Marker for daily consumption of largeamount of alcohol or drugs (barbituates,
phenytoin)
Method: SZAZ- substrate: gammaglutamyl
-nitroanilide
-
7/28/2019 Lecture Enzymes
26/45
Pseudocholinesterase (ChE)
Cholinesterase (true and Pseudo)
Cleaves Acetylcholine: the bodys majorneurotransmitter
True ChE
high activity in the CNS, RBC, lungs and spleen.
Pseudo-ChE or acylcholine acylhydrolase primarily produced in the liver
Also produced by myocardium and pancreas
Important in cleaving SUCCINYLCHOLINE
-
7/28/2019 Lecture Enzymes
27/45
Significant decrease seen after exposure tophosphorus compounds found in insecticide,
nerve gases and pesticide.
Also seen in anesthetic poisoning.
Substrate: acetylcholine
Method:
Michael
Ellman
-
7/28/2019 Lecture Enzymes
28/45
CARDIAC DISORDER PROFILE
CK-MB
AST
LD (LDH) 1 & 2
LD/HBD ratio
MI Biomarkers
-
7/28/2019 Lecture Enzymes
29/45
Creatine Kinase (CK)
found in cardiac, skeletaland brainmuscle
Has 3 isoenzymes:
CK-BB (CK1)= Brain
CK-MB (CK2)= Heart, muscle
CK-MM (CK3)= Muscle, heart
-
7/28/2019 Lecture Enzymes
30/45
Catalyzes
phosphocreatine + ADP creatine + ATP
Methods:
Tanzer-Gilvarg: (forward)
Phospho Kinase + LD Lactate + NAD
Oliver- Rosalki: (reverse)
Hexokinase + G6PD 6-P Gluconate + NADPH
-
7/28/2019 Lecture Enzymes
31/45
ASpartate aminoTransferase
(AST)
Most sensitive enzyme foe skeletal muscle
disease
Collection: AVOID HELOLYSIS
Inhibited by all anticoagulants except heparin(do not use ammonium heparin)
-
7/28/2019 Lecture Enzymes
32/45
Lactate DeHydrogenase 1 & 2
LD 1 (Anodic), while LD 5 is (cathodic)
LD 1 and LD2 (HEAT STABLE)
LD5 (COLD LABILE)
Used in evaluating Cardiac Disorders
-
7/28/2019 Lecture Enzymes
33/45
Normally LD1< LD2
Normal Ratio is 0.5-0.75 or LD2 it is called a flipped ratio Flipped ratio is seen in MI (provided sample is not
hemolyzed)
50% MI: flipped ratio in 48hours 80% MI: flipped ratio in 72 hours
-
7/28/2019 Lecture Enzymes
34/45
LD/HBD(hydroxybutiric
dehydrogenase) Ratio
Normal LD/HBD ratio: 1.2- 1.6
If ratio is 0.8-1.2, MI is suspected
-
7/28/2019 Lecture Enzymes
35/45
Non-Enzymatic MI Markers
MYOGLOBIN Major protein responsible for oxygen supply of
striated muscle
TROPONIN the troponin complex is a component of the thin
filament of striated muscle linked to actin
Three Subunits: Troponin I: an inhibitory subunit
Troponin T: tropomyosin-binding subunit
Troponin C: Calcium-binding subunit
-
7/28/2019 Lecture Enzymes
36/45
ACUTE PANCREATITIS PROFILE
AMYLASE
LIPASE
-
7/28/2019 Lecture Enzymes
37/45
Amylase
found in the SALIVARY GLANDS andPANCREAS
Breaks down starch to simple sugars
Substrate: starch
starch/ amylum maltose glucoseAMS Maltase
-
7/28/2019 Lecture Enzymes
38/45
Substrates:
Pancreatic AMS: diastase
Salivary AMS: ptyalin
Serum AMS is usually pancreatic in origin
microAMS: unbound, free. 50,000 dal. AMS
found in urine
macroAMS: bound to IgG, IgA. High MW.
Measured in serum
-
7/28/2019 Lecture Enzymes
39/45
Methods
SaccharogenicMeasures amount of maltoseproduced (glucose: Somogyi)
Iodometric/amyloclastic Measures starch remaining
ChromogenicMeasures dye released frombreakdown of polysaccharide
Kinetic Method Measures change of NAD toNADH at 340nm
-
7/28/2019 Lecture Enzymes
40/45
Lipase
Breaksdown Triglyceride into fatty acids andglycerol
Significance: Acute Pancreatitis
Substrate: Olive Oil
End Product: Fatty Acids
Methods: Cherry-Crandall
Sigma-Tietz
Titration
-
7/28/2019 Lecture Enzymes
41/45
PROSTATIC CANCER PROFILE
ACP
PSA
-
7/28/2019 Lecture Enzymes
42/45
ACP (pACp)
Optimum pH: 5
Very Labile, Add 5M acetate buffer or citrate
tablet to preserve
ACP are found in Prostate, RBC, bone, liver,kidneys, platelets
Substrate: organic Phosphate such as-glyceroPO4 and -nitrophenylPO4
-
7/28/2019 Lecture Enzymes
43/45
Method
Chemical Inhibition Test
If Total ACP is normal: Stop test
If elevated: suggestive of prostatic CA
do p-ACP by Chemical Inhibition Test
Cu++ Tartrate C4H4O6-2
RBC-ACP Inactivated (+) Unaffected (-)
p-ACP Unaffected (-) Inactivated (+)
-
7/28/2019 Lecture Enzymes
44/45
PSA (Prostate-Specific Antigen)
Member of the kallikrein familyof sereinproteases uniquely produced form theepithelial cells of the prostate gland
Most useful TM for Prostate Cancer
Drawback: weak in distinguishing prostate CA
from nonmalignant prostate lesions
Reference range: 0-4ng/mL
-
7/28/2019 Lecture Enzymes
45/45
Good luck and God bless!
Study well and Pray Harder!